A laser flash-photolysis apparatus was developed to measure the kinetics of carbon monoxide (CO) binding to P450 cytochromes in liver microsomes from rats treated with various drugs and carcinogens. Since numerous forms of P450 contribute to the overall reaction, a difference kinetic method was used to distinguish the kinetic behavior of individual P450s. This method entails analysis of the difference between the kinetic profiles in the absence and presence of a specific P450 effector, and successfully yielded kinetic parameters for individual P450s involved in drug and carcinogen metabolism. Specifically, various polycyclic hydrocarbons differentially accelerated CO binding to the P450 1A1 form, which metabolizes these carcinogens in a size- and shape-dependent manner. In addition to rat- liver microsomes, this approach was applied to a single human P450. Human P450 3A4 was evaluated, and different substrates modulated the CO binding kinetics in a substrate-dependent manner.